Project Summary: Cytotoxic T lymphocytes (CTL?s) are capable of killing tumor cells throughout the body, and can be stimulated to do so by a form of immunotherapy known as PD-1 checkpoint blockade. For unknown reasons, the majority of patients given anti-PD-1 therapeutics do not respond to treatment. Thus, the long-term goal is to identify key factors associated with failure of PD-1 blockade in order to develop strategies that can significantly improve response rates. The overall objective for this specific application is to determine the underlying mechanisms that prevent CTL?s from killing their tumor cell targets, even after anti-PD-1 therapy is on board. NKG7 is a pro- tein expressed by activated T cells which is associated with the membrane of cytotoxic granules. Decreased levels of NKG7 were noted in the CD8+ T cells of patients who did not respond to PD-1 blockade. This leads to the central hypothesis that CD8+ T cells that lack appropriate expression of crucial mediators of cytotoxicity, such as NKG7, are unable to respond to PD-1 blocking therapy. The central hypothesis will be tested by pursu- ing two specific aims: 1) Determine how decreased NKG7 expression levels in CD8+ T cells could lead to fail- ure of anti-PD-1 therapy; and 2) Identify mechanisms that influence the amount of functional NKG7 present in CD8+ T cells. Under the first aim, NKG7 will be knocked-down or over-expressed within CD8+ T cells of both human and mouse origin. These T cells will then be challenged using in vitro and in vivo cytotoxicity assays to determine how modulation of NKG7 levels affects granule release and tumor cell killing by CTL?s. In the sec- ond aim, alternative splicing will be explored as a mechanism that controls the levels of functional NKG7 within CD8+ T cells. RT-PCR will be use to confirm that the relative abundance of aberrant NKG7 mRNA splice vari- ants in the CD8+ T cells of patients at baseline correlates with eventual failure of anti-PD-1 therapy. In vitro as- says will also begin to dissect what upstream signaling may play a role in these splicing changes. The pro- posed research is innovative because it focuses on identifying key factors necessary for the cytotoxic activity of CTL?s, and how defects in this process could lead to failure of PD-1 blockade. The contribution is expected to be significant because it will contribute to the goal of increasing response rates to PD-1 blockade, a therapy with tremendous potential to induce an effective immune attack against a wide range of cancer types and in- crease the survival rate of thousands of patients every year.